Recombinant DNA polymerase for sequencing

Abstract

A recombinant DNA polymerase for sequencing is provided. The recombinant DNA polymerase is a mutant of a 9N° DNA polymerase, the 9N° DNA polymerase mutant can recognize a 3-O-azido-modified reversible terminator and keep high incorporation activity, so that said mutant can be used for second-generation sequencing. The present invention uses a genetically engineered Escherichia coli strain for recombinant expression of the 9N° DNA polymerase mutant, and the expressed mutant enzyme realizes soluble expression in an Escherichia coli system, and therefore has high catalytic activity.

Description

Recombinant DNA polymerase for sequencing Technical Field

[0001] The present invention belongs to the field of biotechnology, and in particular relates to a recombinant DNA polymerase for sequencing. Background Art

[0002] DNA polymerases are used in many reactions involving nucleic acid replication, one important area being DNA sequencing. The current mainstream DNA sequencing method is sequencing-by-synthesis (SBS), which relies on the incorporation and detection of reversible terminators by DNA polymerases to achieve large-scale parallel sequencing of DNA molecules, reducing sequencing costs. Unlike natural nucleotides, the 3-OH group of reversible terminators is modified with a larger reversible group, such as 3-O-allyl, 3-O-(2-nitrobenzyl), or 3-O-azido, and also carries a fluorescent group for easy detection. Due to the differences between the two substrates, natural DNA polymerases often have low inefficiency in incorporating reversible terminators, or even fail to recognize them. Furthermore, the inherent fidelity of DNA polymerases leads to a bias towards the incorporation of modified nucleotides, which can easily lead to phasing in the sequencing reaction and limit the sensitivity of the reaction. On the other hand, sequencing by synthesis only extends one base at a time, and then the reversible terminator 3-OH structure is restored through a chemical reaction and the fluorescent group is removed. However, the linker connected to the fluorescent group still exists on the template DNA. As the sequencing reaction proceeds, the structure of the template DNA and the natural DNA structure become more and more different, resulting in incompatibility between the enzyme and the DNA template. Therefore, a new type of DNA polymerase needs to be modified to overcome this limitation.

[0003] 9N 。 DNA polymerase is a heat-resistant DNA polymerase derived from Thermococcus sp. 9N-7. Since the bacterium was discovered in a volcanic crater on the Pacific Ocean at 9 degrees north latitude, the enzyme was named 9N. 。 DNA polymerase 9N 。 DNA polymerase belongs to the DNA polymerase B family, with a molecular weight of about 95kDa. Like most DNA polymerases, it requires magnesium ions as a cofactor to exert its enzymatic activity and also has 3'-5' exonuclease activity. Its polymerase active center is similar to the right-hand conformation of the Escherichia coli Klenow fragment, which can be further divided into the palm, thumb, and finger domains. An accidental study discovered that 9N from Thermophilus 。 DNA polymerase can well recognize and bind to reversible terminators and can accept a wide range of modified nucleotides as substrates, so B family 9N 。 DNA polymerase is the enzyme of choice for important biotechnologies such as NGS sequencing.

[0004] Therefore, those skilled in the art are committed to 。 DNA polymerase was transformed and recombinantly expressed to produce 9N with low cost suitable for industrial production and high reaction efficiency. 。 DNA polymerase used in optical sequencing.

[0005] Summary of the Invention

[0006] The purpose of the present invention is to provide a recombinant DNA polymerase for sequencing, the recombinant DNA polymerase is 9N 。 DNA polymerase mutants.

[0007] In a first aspect of the present invention, there is provided a 9N 。 DNA polymerase mutant, the 9N 。 The DNA polymerase mutant is mutated at one or more sites selected from the group consisting of position 141, position 143, position 381, position 485, and position 497, wherein the amino acid residues are numbered as shown in SEQ ID NO.2.

[0008] In another preferred embodiment, the 9N 。 The amino acid sequence of the DNA polymerase mutant has at least 80% homology to SEQ ID NO.2; more preferably, at least 90% homology; most preferably, at least 95% homology; such as at least 96%, 97%, 98%, 99% homology.

[0009] In another preferred embodiment, the 9N 。 The number of mutation sites in the DNA polymerase mutant is 1-5, preferably 4 or 5.

[0010] In another preferred embodiment, the 9N 。 The DNA polymerase mutant is 9N shown in SEQ ID NO.2 。 The amino acid residues at positions 141, 143, 381, 485 and 497 of the DNA polymerase mutated; in addition, the amino acid residue at position 141 mutated to A, the amino acid residue at position 143 mutated to A, the amino acid residue at position 381 mutated to K, the amino acid residue at position 485 mutated to L, and the amino acid residue at position 497 mutated to V.

[0011] In another preferred embodiment, the 9N 。 The amino acid sequence of the DNA polymerase mutant is shown in SEQ ID NO.4.

[0012] The second aspect of the present invention provides a polynucleotide molecule encoding the 9N 。DNA polymerase mutants.

[0013] The third aspect of the present invention provides a vector, wherein the vector contains the nucleic acid molecule described in the second aspect of the present invention.

[0014] The fourth aspect of the present invention provides a host cell, wherein the host cell contains the vector described in the first aspect of the present invention or the chromosome is integrated with the nucleic acid molecule described in the second aspect of the present invention.

[0015] In another preferred embodiment, the host cell is a prokaryotic cell or a eukaryotic cell.

[0016] In another preferred embodiment, the prokaryotic cell is Escherichia coli.

[0017] In another preferred embodiment, the eukaryotic cell is a yeast cell.

[0018] The fifth aspect of the present invention provides a method for preparing the 9N 。 A method for producing a DNA polymerase mutant, comprising the steps of:

[0019] (i) culturing the host cell according to the fourth aspect of the present invention under suitable conditions to express the 9N 。 DNA polymerase mutants; and

[0020] (ii) Separation of the 9N 。 DNA polymerase mutants.

[0021] In another preferred embodiment, the temperature for culturing the host cells in step (i) is 20°C-40°C; preferably 25°C-37°C, such as 35°C.

[0022] In another preferred embodiment, the host cell in step (i) is an Escherichia coli cell.

[0023] In a sixth aspect of the present invention, a kit is provided, wherein the kit comprises the 9N 。 DNA polymerase mutants.

[0024] The seventh aspect of the present invention provides the 9N described in the first aspect of the present invention. 。 DNA polymerase mutants are used in the preparation of gene sequencing kits.

[0025] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features described in detail below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be listed here one by one. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 shows 9N 。 DNA polymerase expression test results;

[0027] Figure 2 is a standard curve diagram for enzyme activity detection;

[0028] Figure 3 is an electrophoresis diagram of the commercial enzyme substrate compatibility test;

[0029] FIG4 is an electrophoresis diagram of substrate compatibility test of a preferred mutant enzyme of the present invention. DETAILED DESCRIPTION

[0030] The inventors have conducted extensive and in-depth research and used genetically engineered Escherichia coli to recombinantly fuse 9N 。 DNA polymerase mutant, expressing 9N 。 The DNA polymerase mutant has achieved soluble expression in the E. coli system, with high protein yield and high catalytic activity. It has the advantages of short production cycle, easy purification of expression products, low cost, etc., and can achieve 9N 。 The present invention was developed based on the industrial production of a DNA polymerase mutant. This mutant enzyme can recognize a 3-O-azido-modified reversible terminator and maintain high incorporation activity, making it applicable to next-generation sequencing.

[0031] Before describing the present invention, it should be understood that the present invention is not limited to the specific methods and experimental conditions described, because such methods and conditions may vary. It should also be understood that the terminology used herein is intended to describe specific embodiments only and is not intended to be limiting, and the scope of the present invention will be limited only by the appended claims.

[0032] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. As used herein, when used in reference to a specific recited value, the term "about" means that the value may vary by no more than 1% from the recited value. For example, as used herein, the expression "about 100" includes all values ​​between 99 and 101 (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0033] Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described.

[0034] Currently 9N 。 DNA polymerase mutants have been commercialized (named Therminator TMA DNA polymerase (DNA polymerase) carries three mutations: D141A, E143A, and A485L. The D141A and E143A mutations are located in the 3'-5' exonuclease domain, resulting in a loss of proofreading activity. The A485 amino acid is located in the finger domain, facing away from the polymerase active center and preventing direct contact with substrate nucleotides. Therefore, mutations at this site may reduce the enzyme's ability to discriminate for modified nucleotides, but the enzyme's optimal substrates are ddNTPs and acyclic nucleotides (AcyNTPs). Another mutant of the enzyme, in addition to the three aforementioned mutations, also carries the Y409A mutation, enabling recognition and incorporation of reversible terminators. However, this polymerase mutant has low incorporation efficiency for 3'-modified reversible terminators, requiring longer incubation times and higher concentrations of the 3'-modified reversible terminator to complete the reaction.

[0035] In a preferred embodiment of the present invention, wild type 9N 。 The base sequence of DNA polymerase (optimized for synonymous codon preference in Escherichia coli, SEQ ID NO. 1):

[0036] In a preferred embodiment of the present invention, wild type 9N 。 The DNA polymerase amino acid sequence (SEQ ID NO.2) is as follows:

[0037] According to the three-dimensional structure published in the database (PDB: 5omv), 9N was characterized by molecular simulation technology. 。 DNA polymerase undergoes amino acid modification. The relevant mutation sites include:

[0038] D141, E143, T267, V282, R381, L408, Y409, P410, K464, Q483, A485, N491, Y494, Y497, E578, E580, the amino acid residue numbering is according to SEQ ID NO. 2.

[0039] The final test results showed that the mutant containing the combination of D141A, E143A, R381K, A485L, and Y497V mutations had the best performance in integrating fluorescently modified sequencing substrates. 。 The base sequence of DNA polymerase (optimized for synonymous codon preference in E. coli, SEQ ID NO. 3) is as follows:

[0040] In a preferred embodiment of the present invention, the mutant 9N 。The sequence of DNA polymerase is as follows (SEQ ID NO.4):

[0041] Those skilled in the art can obtain the mutant enzyme gene sequence of the present invention using conventional methods, such as complete artificial synthesis or PCR synthesis. A preferred synthesis method is asymmetric PCR. Asymmetric PCR uses unequal amounts of a pair of primers to produce a large amount of single-stranded DNA (ssDNA) after PCR amplification. These primers are respectively referred to as the non-restricting primer and the restricting primer, and their ratio is generally 50-100:1. In the first 10-15 cycles of the PCR reaction, the amplified product is primarily double-stranded DNA. However, once the restricting primer (low-concentration primer) is consumed, PCR guided by the non-restricting primer (high-concentration primer) will produce a large amount of single-stranded DNA. Primers used for PCR can be appropriately selected based on the sequence information of the present invention disclosed herein and can be synthesized using conventional methods. The amplified DNA / RNA fragments can be separated and purified using conventional methods, such as gel electrophoresis.

[0042] The mutant enzyme of the present invention can be expressed or produced by conventional recombinant DNA technology, comprising the steps of:

[0043] (1) transforming or transducing a suitable host cell with a polynucleotide encoding the protein of the present invention, or with a recombinant expression vector containing the polynucleotide;

[0044] (2) culturing the host cells in a suitable culture medium;

[0045] (3) Isolate and purify the target protein from the culture medium or cells to obtain the target enzyme.

[0046] Methods well known to those skilled in the art can be used to construct expression vectors containing the DNA sequence encoding the enzyme of the present invention and appropriate transcriptional / translational control signals, preferably the commercially available vector pET28. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombination techniques, and the like. The DNA sequence can be operably linked to an appropriate promoter in the expression vector to direct mRNA synthesis. The expression vector also includes a ribosome binding site for translation initiation and a transcriptional terminator. In addition, the expression vector preferably contains one or more selectable marker genes to provide a phenotypic trait for selection of transformed host cells.

[0047] The recombinant vector comprises, in the 5' to 3' direction: a promoter, a target gene, and a terminator. If necessary, the recombinant vector may further comprise the following elements: a protein purification tag; a 3' polyadenylation signal; a non-translated nucleic acid sequence; a transport and targeting nucleic acid sequence; a selectable marker (antibiotic resistance gene, fluorescent protein, etc.); an enhancer; or an operator.

[0048] Methods for preparing recombinant vectors are well known to those of ordinary skill in the art. Expression vectors can be bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses, or other vectors. In short, any plasmid or vector can be used as long as it can replicate and be stable in the host.

[0049] Those skilled in the art can construct vectors containing the promoter of the present invention and / or target gene sequence using well-known methods, including in vitro recombinant DNA technology, DNA synthesis technology, in vivo recombination technology, etc.

[0050] The expression vectors of the present invention can be used to transform appropriate host cells to cause the host to transcribe the target RNA or express the target protein. The host cells can be prokaryotes such as Escherichia coli, Corynebacterium glutamicum, Brevibacterium flavum, Streptomyces, or Agrobacterium; lower eukaryotic cells such as yeast cells; or higher eukaryotic cells such as plant cells. Those skilled in the art will appreciate how to select appropriate vectors and host cells. Transformation of host cells with recombinant DNA can be performed using conventional techniques familiar to those skilled in the art. When the host is a prokaryotic organism (such as Escherichia coli), CaCl2 treatment or electroporation can be used. When the host is a eukaryotic organism, the following DNA transfection methods can be used: calcium phosphate coprecipitation, conventional mechanical methods (such as microinjection, electroporation, liposome packaging, etc.). Plants can also be transformed using methods such as Agrobacterium transformation or gene gun transformation, for example, the leaf disc method, the embryo transformation method, and the flower bud immersion method. Transformed plant cells, tissues, or organs can be regenerated into plants using conventional methods to obtain transgenic plants.

[0051] The term "operably linked" means that the target gene to be transcribed and expressed is linked to its control sequence in a conventional manner in the art so as to be expressed.

[0052] Cultivation of engineered bacteria and fermentation production of target protein

[0053] After obtaining the engineered cells, the engineered cells can be cultured under suitable conditions to express the protein encoded by the gene sequence of the present invention. Depending on the host cell, the culture medium used can be selected from various conventional culture media, and the cells are cultured under conditions suitable for the growth of the host cells. After the host cells have grown to an appropriate cell density, the selected promoter is induced using a suitable method (such as temperature shift or chemical induction), and the cells are cultured for an additional period of time.

[0054] In the present invention, conventional fermentation conditions can be used. Representative conditions include (but are not limited to):

[0055] (a) Regarding temperature, the fermentation and induction temperature of the enzyme is maintained at 25-37°C;

[0056] (b) With regard to the pH value during the induction period, the pH during the induction period is controlled at 3-9;

[0057] (c) Dissolved oxygen (DO) is controlled at 10-90%. This can be maintained by introducing an oxygen / air mixture.

[0058] (d) As for feed, feed types should preferably include carbon sources such as glycerol, methanol, and glucose, which can be fed individually or in combination;

[0059] (e) With respect to the IPTG concentration during the induction period, conventional induction concentrations can be used in the present invention, and the IPTG concentration is usually controlled at 0.1-1.5 mM;

[0060] (f) The induction time is not particularly limited, but is usually 2 to 20 hours, preferably 5 to 15 hours.

[0061] The target protein of the present invention is present intracellularly in Escherichia coli cells. The host cells are collected by centrifugation and then disrupted by high pressure, mechanical force, enzymatic cell lysis, or other cell disruption methods to release the recombinant protein. High pressure is preferred. The host cell lysate can be initially purified by methods such as flocculation, salting out, and ultrafiltration, followed by purification by chromatography, ultrafiltration, or directly by chromatography.

[0062] Chromatographic techniques include cation exchange chromatography, anion exchange chromatography, gel filtration chromatography, hydrophobic chromatography, affinity chromatography, etc. Commonly used chromatography methods include:

[0063] 1. Anion exchange chromatography:

[0064] Anion exchange chromatography media include (but are not limited to) Q-Sepharose and DEAE-Sepharose. If the fermentation sample has a high salt concentration that affects binding to the ion exchange media, the salt concentration should be reduced before ion exchange chromatography. The sample can be equilibrated with a buffer exchanger using dilution, ultrafiltration, dialysis, gel filtration chromatography, or other methods until the equilibration buffer system matches that of the corresponding ion exchange column. The sample can then be loaded and eluted using a salt concentration or pH gradient.

[0065] 2. Hydrophobic chromatography:

[0066] Hydrophobic chromatography media include (but are not limited to) phenyl-sepharose, butyl-sepharose, and octyl-sepharose. The sample is loaded with a solution containing a salt concentration increased by adding NaCl, (NH₄)₂SO₄, or other methods. Elution is then performed by decreasing the salt concentration. Hydrophobic chromatography removes contaminating proteins with significantly different hydrophobicities.

[0067] 3. Gel Filtration Chromatography

[0068] Hydrophobic chromatography media include (but are not limited to): Sephacryl, Superdex, Sephadex. Gel filtration chromatography can be used to replace the buffer system or further purify the product.

[0069] 4. Affinity chromatography

[0070] Affinity chromatography media include (but are not limited to): HiTrap TM HeparinHPColumns.

[0071] 5. Membrane filtration

[0072] Ultrafiltration media include organic membranes such as polysulfone membranes, inorganic membranes such as ceramic membranes, and metal membranes. Purification and concentration can be achieved through membrane filtration.

[0073] The main advantages of the present invention are:

[0074] (1) 9N of the present invention 。 The DNA polymerase mutant can recognize 3-O-azide-modified reversible terminators and maintain high incorporation activity, and can be applied in optical sequencing.

[0075] (2) 9N of the present invention 。 The DNA polymerase mutant can be expressed in large quantities in an E. coli expression system in a soluble form, is easy to purify with a high yield, and maintains a high biocatalytic activity.

[0076] The present invention will be further described in detail below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the present invention. The experimental methods in the following examples, for which detailed conditions are not specified, are generally performed according to conventional conditions such as those described in "Molecular Cloning Laboratory Manual" by Sambrook.J et al. (translated by Huang Peitang et al., Beijing: Science Press, 2002), or according to the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are calculated by weight. The experimental materials and reagents used in the following examples can be obtained from commercial sources unless otherwise specified.

[0077] Example 1 Escherichia coli 9N 。 Construction, expression and purification of DNA polymerase plasmid

[0078] 1) Using wild type 9N 。Based on the protein sequence of DNA polymerase (SEQ ID NO. 2) and in combination with the experimental design requirements of the present invention, the coding base sequences of the wild type and different mutants were obtained after optimization of the synonymous codon preference of Escherichia coli. Each base sequence was ligated into the vector pET-28a(+) and fused to the C-terminus with a (His)6 tag. It was synthesized by Suzhou Jinweizhi Biotechnology Co., Ltd.

[0079] Table 1

[0080] 2) Transformation of recombinant plasmid into Escherichia coli BL21(DE3)

[0081] Take 1 μL of plasmid and add it to 30 μL of competent E. coli BL21(DE3) on ice. Place the culture on ice for 20 minutes, heat shock the culture in a 42°C water bath for 45 seconds, and immediately place it on ice for 2 minutes. Add 400 μL of antibiotic-free SOC medium and incubate the culture at 37°C with shaking at 220 rpm for 50 minutes. Spread 100 μL of the culture evenly onto an LB plate containing 100 μg / mL kanamycin and incubate the culture overnight at 37°C.

[0082] 3) Target protein expression

[0083] Pick the single clone from step 2) and inoculate it in SOC medium containing 100 μg / mL kanamycin aseptically, and culture it at 37°C with shaking at 220 rpm until OD 600 At a concentration between 0.6 and 0.8, IPTG was induced (final concentration was 0.1 mM), and the cells were cultured at 18°C ​​overnight with shaking. The group without IPTG was used as a control, and then cultured at 37°C for 3 hours. Each experiment was repeated once. The samples were ultrasonically broken and identified by SDS-PAGE. The results showed that 9N could be achieved in the culture medium of strains 4, 5, 6, 7, and 8. 。 DNA polymerase was expressed soluble in the supernatant, and strain 8 produced higher yields of the soluble target protein than the other strains. No soluble target protein was detected in strains 2 and 3. The target protein had a molecular weight of approximately 100 kDa, which was consistent with the protein size predicted on the Expasy website (95 kDa) (Figure 1).

[0084] 4) 9N 。 Purification of DNA polymerase

[0085] The solution formula used to prepare the samples is as follows:

[0086] Buffer A: 50mM Tris, 50mM NaCl, 5% Glycerol, pH 8.0

[0087] Buffer B: 50mM Tris, 50mM NaCl, 500mM Imidazole, 5% Glycerol, pH 8.0

[0088] Buffer C: 100mM Tris, 1M NaCl, 10% Glycerol, pH 8.0

[0089] Lysis Buffer: 50mM Tris, 300mM NaCl, 5% Glycerol, pH 8.0

[0090] 9N dialysis buffer: 10 mM Tris-HCl, 100 mM KCl, 1 mM DTT, 0.1 mM EDTA, 50% Glycerol, pH 7.4

[0091] The specific steps are as follows:

[0092] 1.5 L of bacterial liquid was cultured in a shake flask with SOC medium. The expression conditions were consistent with those for the expression of the target protein in 3). The bacteria were collected by centrifugation. About 20 g of bacteria were weighed, and 100 ml of Lysis Buffer was added and resuspended on ice. Ultrasonic cell disruption: Φ10 probe, power 20%, working for 5.5 seconds, stopping for 9.9 seconds, and ultrasonic disruption for 30 minutes. After disruption, the sample was placed in a 70°C water bath for heat treatment for 20 minutes. Centrifuge at 20,000 rpm and 4°C for 35 minutes, take the supernatant, and filter through a 0.22 μm membrane. The supernatant was subjected to Ni-column affinity chromatography, and the purification column used was HisTrap TM HP, 0-60% Buffer B for linear elution, take the main peak eluate for ion exchange elution, the purification column used is HisTrap TM SP-HP was linearly eluted with 0-60% Buffer C to obtain 90 ml of target protein solution.

[0093] The purified sample was dialyzed in dialysate overnight, and the protein concentration was determined by SDS-PAGE grayscale analysis.

[0094] The protein concentrations of the purified samples are shown in Table 2.

[0095] Table 2

[0096] The results showed that the protein concentration and protein yield of the sample purified from strain 8 were the highest.

[0097] Example 2 Recombinant 9N 。 DNA polymerase activity assay

[0098] 1. Experimental Purpose

[0099] Calibrated 9N 。 DNA polymerase activity.

[0100] 2. Experimental Materials

[0101] Sample: 9N 。 DNA polymerase

[0102] Equipment and reagents: Real-time fluorescence quantitative PCR system, Picogreen, λ DNA (Merck).

[0103] Substrate T2 (SEQ ID NO. 5):

[0104] The above substrate was synthesized, purified by HPLC, and prepared into a 100 pmol / μL mother solution according to experimental requirements.

[0105] 4) 9N 。 Preparation of enzyme dilution:

[0106] 10mM Tris-HCl, 100mM KCl, 1mM DTT, 0.1mM EDTA, 50% Glycerol, (pH 7.4@25℃)

[0107] 3. Experimental Procedure

[0108] 3.1 Prepare 10× PCR Buffer according to the following table:

[0109] 3.2 Prepare the λDNA pre-staining solution according to the following table:

[0110] 3.3 λDNA was diluted into gradient according to the following table:

[0111] 3.4 Dilution of test samples:

[0112] 9N 。 Enzyme samples were treated with 9N 。 Dilute with enzyme diluent. Dilution multiples are 1 / 100, 1 / 200, 1 / 400, 1 / 800, 1 / 1600, and 1 / 3200. The dilution multiple can be adjusted according to actual conditions and the assay can be performed at the same protein concentration.

[0113] 3.5 Reaction system configuration (single reaction system)

[0114] Each dilution gradient of λDNA and λDNA pre-staining solution was mixed evenly in a ratio of 1:1. The 0ug / ml λDNA group was used as the control group. After calculating the required number of wells (N), the above reaction system was divided by 9N with N+3. 。 Mix the components outside the enzyme evenly and add 18.7 μL per well into a 96-well plate. Add 20 μL of each gradient of λ DNA per well into a 96-well plate. Repeat at least 3 times for each concentration gradient of sample enzyme and λ DNA. Then add the diluted 9N 。 Enzyme (operate on ice), 1.3 μL / well, shake and centrifuge, NTC group add 1.3 μL 9N 。 Enzyme diluent. For the λDNA group, no sample enzyme was added. Place the 96-well plate in a qPCR instrument and run the SYBR read at 74°C for 2 seconds (74°C for 14 seconds) for 120 cycles, then read the SYBR buffer at 74°C for 2 seconds, monitoring fluorescence in real time.

[0115] 4. Experimental results:

[0116] 4.1 Plotting the λDNA standard curve

[0117] 1) Export the Q5 data to an Excel spreadsheet, select the fluorescence values ​​of the λDNA standard and λDNA NTC groups at the 30th cycle, calculate the average of the three groups of data at each λDNA concentration, and subtract the average of the three groups of data from the NTC group to obtain the net fluorescence value. Use the amount of λDNA input as the horizontal axis and the net fluorescence value as the vertical axis to draw a linear standard curve with R 2 >0.99, the standard curve is shown in Figure 2:

[0118] Export the Q5 machine data to an Excel table, select each gradient 9N 。 The fluorescence values ​​of the enzyme experimental group and the control group NTC group at the 30th cycle were calculated for each gradient 9N 。 The net fluorescence value was obtained by subtracting the average of the three NTC groups from the average of the three enzyme groups. The net fluorescence value was then inserted into the λDNA standard curve to calculate the amount of DNA generated, A1. A1 / 649 was used to calculate the amount of dNTPs consumed in the reaction, A2, in nmol. Finally, the 9N value was calculated using the formula A2*0.449*dilution factor. 。 Enzyme activity.

[0119] Enzyme activity definition: 1 U of enzyme is the amount of enzyme required to incorporate 10 nmol of dNTPs at 74°C in 30 min.

[0120] The measurement results are shown in Table 3 below.

[0121] Table 3 Note: The commercial enzyme is Therminator TMDNA polymerase (NEB).

[0122] Results: λDNA concentration standard curve R 2 >0.99, strain 8 and strain 6 had higher activities, about 107U / μL and 53U / μL respectively. According to the results of the inter-batch enzyme test, when the CV value was within 10% and the amount of dNTPs consumed was 0.01-0.14nnmol, 9N 。 The enzyme activity results at each gradient interval were similar. The activity of the enzyme of the present invention was much higher than that of the commercial enzyme (Therminator DNA polymerase).

[0123] Example 3 Recombinant 9N 。 DNA polymerase substrate compatibility test

[0124] 1. Experimental Purpose

[0125] Test 9N 。 DNA polymerase performance

[0126] 2. Experimental Materials

[0127] 1) Sample: 10X Reaction Buffer (NEB), 9N each 。 DNA polymerase mutants.

[0128] 2) Equipment: Chemiluminescence fluorescence imaging system, gene analyzer

[0129] 3) Substrate:

[0130] 9N Test-A (SEQ ID NO. 6):

[0131] 9N Test-T (SEQ ID NO. 7):

[0132] 9N Test-G (SEQ ID NO. 8):

[0133] 9N Test-C (SEQ ID NO. 9):

[0134] The sequencing substrates contain four nucleotides: A, T, G, and C. Their 3'-end OH groups are replaced with azide groups and each carries a fluorescent group. A bases fluoresce in both the red and green channels, T bases fluoresce in the green channel, C bases fluoresce in the red channel, and G bases do not fluoresce in either red or green channel. These four templates were synthesized outsourced (HPLC purified, purity >90%) and added to sterile water to a final concentration of 100 pmol / μL stock solution. The stock solution was then diluted 10-fold with sterile water to a working concentration of 10 pmol / μL.

[0135] 4) Experimental Materials

[0136] 10X sequencing substrate, GeneScan 600LIZ Size Standard v2.0, HiDi-Formamide deionized formamide, 6X DNA Loading Buffer.

[0137] 3. Experimental steps

[0138] 3.1 Substrate compatibility test

[0139] 1) Reaction Buffer Configuration

[0140] 10X sequencing substrate, 10X Reaction Buffer and deionized water were prepared into a 1.1X substrate concentration mix, and 9N 。 Enzyme: 1.1X Mix at a ratio of 1:9, enzyme concentration is 5 U / μL.

[0141] 2) Reaction system configuration (single-person reaction):

[0142] Calculate the required number of wells (N), and use N+3 as the number, and apply 9μL per well to 8 strips, and then add the annealed 9N 。 Test-A / T / G / C, 1 μL / well, shake, and centrifuge. Place in a PCR instrument and incubate at 60°C for 4 min and 95°C for 10 min. Add 2 μL of 6X DNA Loading Buffer (dye-free) to the reaction product, with a final concentration of 0.1% SDS, and perform 15% Urea-PAGE electrophoresis. Scan the gel using a chemiluminescence fluorescence imaging system in both the red and green channels.

[0143] 4 Experimental results

[0144] The test results showed that the enzyme samples from strains 1, 5, and 6, as well as the commercial enzyme sample, were incompatible with the sequencing substrate, with no single-base extension band observed at the 100-bp position. The enzyme sample from strain 7 exhibited a very weak single-base extension band signal at the 100-bp position, indicating low mutant incorporation efficiency. Representative electropherograms of the commercial enzyme substrate compatibility test are shown in Figure 3. As can be seen in the figure, no single-base extension band was observed at the 100-bp position, whether imaged using red or green light, indicating that the commercial enzyme sample was incompatible with the sequencing substrate.

[0145] Strain 8 9N 。 The electrophoresis diagram of the DNA polymerase sample substrate compatibility test is shown in Figure 4. The red light channel scan shows the A template and C template group, and a clear single base extension band appears at 100 bp; the green light channel scan shows the A template and T template group, and a clear single base extension band appears at 100 bp. 。 DNA polymerase mutants are compatible with sequencing substrates.

[0146] All documents mentioned in this application are incorporated herein by reference, just as if each document were incorporated herein by reference individually. It should also be understood that after reading the above teachings of the present invention, those skilled in the art may make various changes or modifications to the present invention, and that such equivalents also fall within the scope of the claims appended hereto.

Claims

1. A 9N° DNA polymerase mutant, characterized in that: The amino acid sequence of the 9N° DNA polymerase mutant is shown in SEQ ID NO.

4.

2. A polynucleotide molecule, characterized in that The polynucleotide molecule encodes the 9N° DNA polymerase mutant described in claim 1.

3. A carrier, characterized in that The vector contains the nucleic acid molecule according to claim 2.

4. A host cell, characterized in that The host cell contains the vector of claim 3 or the nucleic acid molecule of claim 2 is integrated into the chromosome.

5. The host cell according to claim 4, characterized in that The host cell is a prokaryotic cell or a eukaryotic cell.

6. The host cell according to claim 5, characterized in that The prokaryotic cells are Escherichia coli cells.

7. A method for preparing the mutant 9N° DNA polymerase according to claim 1, characterized in that: Includes steps: (i) culturing the host cell of claim 4 under suitable conditions to express the 9N° DNA polymerase mutant; and (ii) isolating the 9N° DNA polymerase mutant.

8. The method according to claim 7, characterized in that The host cell in step (i) is an Escherichia coli cell.

9. A kit, characterized in that: The kit comprises the 9N° DNA polymerase mutant according to claim 1.

10. Use of the 9N° DNA polymerase mutant according to claim 1 in preparing a gene sequencing kit.

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